ES2361007T3 - HANDLING OF THE GASTROINTESTINAL TRANSIT RATE WHEN MODULATING THE INTESTINAL METHANE CONCENTRATION. - Google Patents

Abstract

The present invention relates to a method of diagnosing constipation in human subjects that comprises detecting the presence of methane in a breath sample from the subject. The invention also concerns agents that may be used to manipulate the rate of gastrointestinal transit and thereby treat conditions such as constipation and diarrhea.

Description

BACKGROUND OF THE INVENTION

Related Technique Discussion

5 Irritable bowel syndrome (IBS) is a common gastrointestinal disorder seen in more than 15% of the population (1,2).

During the last years, there has been progress in the characterization of irritable bowel syndrome (IBS). Studies have shown altered motility of the intestine (3), peripheral (4) and central (5) sensory dysfunction, as well as an exaggerated response to tension (6) in this syndrome. However, there is no finding that

10 can identify in the majority of patients and by extension, there is no diagnostic test associated with IBS. As a result, researchers have created a complex diagnostic scheme such as the Rome criteria to help diagnose and categorize the syndrome (7,8).

A consistent clinical finding in IBS is gas in combination with swelling and visible distention (9,10). Koide et al. They recently found that gas from the small intestine has increased significantly in IBS

15 compared to controls (11) independent of whether the subjects form subgroups with diarrhea, constipation or pain.

Excessive gas in the small intestine can occur as a result of increased gas production within the intestine by bacterial fermentation. Hydrogen and methane are common gases excreted during the breath test (43). Although hydrogen production seems more ubiquitous, methane production 20 is seen in 36-50% of healthy subjects (27, 41, 42). In particular, it is observed that methane is common in diverticulosis (25), and is less prevalent in diarrheal conditions such as Crohn's colitis or ulcerative colitis (2628). Recent data suggest that children with encopresis have excessive methane breath in the lactulose breath test ("LBT"; 13). This finding has not been extended to adults with predominant IBS constipation.

A condition known to produce excessive small intestine gas is bacterial ascending growth of the

25 small intestine (SIBO). Bacterial ascending growth of the small intestine is a condition in which the small intestine is colonized by excessive amounts of the flora of the upper or lower gastrointestinal tract. Although there are many conditions associated with SIBO, recent studies have shown an increased prevalence of SIBO in irritable bowel syndrome (IBS) (12) and this is a recognized cause of diarrhea in inflammatory bowel disease (IBD) (28, 39, 40).

30 There is some support for the association between altered breath test results and enteric flora in IBS. In one study, it is found that 56% of predominant IBS subjects with diarrhea have a positive 13C-xylose breath test (20). In another study, flagil is reported to be superior to placebo since it reduces clinical symptoms in IBS (21). The authors in that document have uncertainty about the mechanism for this improvement.

35 One method to diagnose SIBO is the lactulose breath test (LBT) where ascending overgrowth is considered to be present if an increase of more than 20 ppm in the concentration of hydrogen or methane in breath is observed within 90 minutes after oral administration of lactulose (19).

In a recent study, we suggest that a large percentage (78%) of IBS subjects have SIBO when they are diagnosed by the lactulose breath test (12). Some workers criticize the reliability of the LBT to diagnose 40 SIBO because in the identification of the infectious process, the culture is the golden standard. The main fact with cultivation is accessibility. Riordan, et al. compare the breath test to direct the crop and find that the breath test lacks reliability (29). This and other similar studies are confused by their selection of subjects who have surgically altered anatomical predisposition for the development of the upper GI tube. SIBO Because SIBO (in surgically neophyte patients) is often an expansion of bacteria

45 colonic, the direction of expansion is retrograde first involving the distal small intestine. As such, direct culture is only practical in the patient whose SIBO is very severe that bacteria have expanded shortly within the duodenum or proximal jejunum.

Regardless of some skepticism about the reliability of LBT to diagnose SIBO, there are similarities between SIBO and IBS. Inflammation, a characteristic of SIBO, is also classically associated with IBS 50 (10). In SIBO, inflammation is due to the fermentation of nutrients in the small intestine. Recently, gas studies at IBS have been limited to flatulence research. Still, these studies still suggest the

presence of excessive bacteria in IBS. King, et al found that hydrogen production by IBS subjects is five times higher which implies excessive enteric bacteria (22). Recently, data suggests that IBS patients have excessive gas and that this gas is located in the small intestine (11). However, the predominant subgroups of contrast diarrhea and constipation in IBS remain unexplained.

The speed of transit through the small intestine is normally regulated by the inhibitory mechanisms located in the distal and proximal small intestine known as the jejunum valve and the ileal valve. Inhibitory feedback is activated to delay transit when digestion of final products makes contact with nutrient sensors of the small intestine, (For example, Lin, HC, U.S. Patent No. 5,977,175; Dobson, CL et al., The effect of oleic acid on the human ileal brake and its implications for small intestinal transit of tablet formulations, Pharm. Res. 16 (1): 92-96 [1999]; Lin, HC et al., Intestinal transit is more potently inhibited by fat in the distal (Ileal brake) than in the proximal (jejunal brake) gut, Dig. Dis. Sci. 42 (1): 19-25 [1997]; Lin, HC et al., Jejunal brake: inhibition of intestinal transit by fat in the proximal small intestine, Dig. Dis. Sci, 41 (2): 326-29 [1996a]).

Never before has methane been reported in the intestinal lumen that affects the gastrointestinal transit rate.

GB 423 083 is related to the use of Saccharomyces to treat constipation. GB 2 338 244 is related to Lactobacilli and Bifidobacteria to treat diarrhea and constipation. JP 08 310960 relates to Lactobacillus as a purgative. EP 1 609 852 describes Bifidobacteria to treat constipation. JP 60 133852 describes fructo-oligosaccharides to treat constipation.

SUMMARY OF THE INVENTION

A first aspect of the invention provides a selective methanogenic inhibitor, selected from monensin and an HMG-CoA reductase inhibitor, for use in the treatment of constipation.

A further aspect of the invention provides an agent comprising methane or a methane precursor that causes the partial pressure of methane in the intestine of a subject being treated to be increased for use in the treatment of diarrhea. An embodiment of this aspect of the invention is where the agent is a methane precursor comprising a catalyst and chemical substrate that causes the partial pressure of methane in the intestine of a subject to be treated to increase.

The present invention relates to a method for manipulating the gastrointestinal transit rate in a mammalian subject, which includes a human patient. The method involves: (a) increasing the rate of gastrointestinal transit by causing partial pressure of methane in the intestine of the subject to be reduced; and (b) reduce the rate of gastrointestinal transit by causing partial pressure of methane in the intestine of the subject, for example in the distal intestine, which is going to increase.

Thus, by practicing the method of the invention to increase the rate of gastrointestinal transit, constipation and disorders that present constipation can be treated in subjects in whom abnormally high levels of intestinal methane are detectable (for example, in cases of constipation-syndrome predominant irritable bowel [IBS], pseudo-obstruction, colonic inertia, postoperative ileus, encopresis, liver disease, medication-induced constipation or encephalopathy). According to this description, the partial pressure of methane in the subject's intestine can be reduced by administering to the subject's intestinal lumen a selective inhibitor of methanogeny, such as monensin, or a probiotic agent that displaces the methanogen, or a prebiotic agent that It inhibits the growth of methanogenic bacteria or promotes the growth of competent non-methanogenic intestinal flora.

Subsequently, we also describe the use in the manufacture of a drug for the treatment of constipation, a selective methanogen inhibitor, or a probiotic agent that displaces the methanogen, or a prebiotic agent that inhibits the growth of methanogenic bacteria or promotes the competent non-methanogenic intestinal flora growth.

And alternatively, by practicing the method of the invention to reduce the rate of gastrointestinal transit, patients with diarrhea and diarrhea disorders (eg, cases of predominant IBS diarrhea, Crohn's disease, ulcerative colitis, celiac disease) can be treated. , microscopic colitis, rapid gastric evacuation syndrome, rapid transit, small intestine syndrome, post-gastrectomy syndrome, diabetic diarrhea, hyperemesis, or antibiotic-associated diarrhea). According to this description, the partial pressure of methane in the subject's intestine can be increased by administering methane gas to the subject's intestinal lumen, for example in the distal segment of the subject's intestine, or by administering to the subject a methanogenic probiotic agent or probiotic agent that improves methanogenesis.

Subsequently, we also describe the use in the manufacture of a drug for the treatment of diarrhea, methane or a methane precursor, or a methanogenic prebiotic agent or other prebiotic agent that improves methane, or a prebiotic agent that improves methanogenesis. .

These and other advantages and features of the present invention will be more fully described in a detailed description of the preferred embodiments that follow.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows a patient flow chart for a double-blind, placebo-controlled study that confirms that an abnormal lactulose breath test is more prevalent in IBS than normal controls, and that antibiotic treatment in IBS that leads to improvement. in the symptoms and that is based on the antibiotic-induced normalization of the breath test.

Figure 2 shows the percentage of improvement in the composite classification based on treatment and success in normalizing LHBT. Data =% average reduction in composite classification; the difference in the composite classification is significant (p-0.01, 1-way ANOVA). The difference in the reported improvement of the patient is also significant (p <0.000001, one-way ANOVA). In the groups treated with neomycin, the data is analyzed according to the success of the treatment. Neo = Neomycin.

Figure 3 shows a comparison of the reported percentage of bowel normalization between and within gender groups; NS = not significant.

Figure 4 shows the pattern of gas production with the type of IBS symptom, that is, predominant IBS constipation (non-shaded bars; p <0.00001) versus predominant IBS diarrhea (shaded bars; p <0.001).

Figure 5 shows the pattern of gas production in IBS patients (n = 65) with respect to symptom severity in those with predominant IBS constipation (non-shaded bars; p <0.00001) versus those with predominant IBS diarrhea (bars shaded; p <0.00001).

Figure 6 illustrates the effect on intestinal transit in dogs administered with 180 ml of ambient air (circles) or methane gas (squares) by bolus delivery to the distal intestine. Slower transit methane.

Figure 7 shows the classification of diarrhea severity and mean constipation of all subjects (n = 551) with SIBO as a function of the type of gas pattern produced in LBT; p <0.00001 for a tendency to reduce diarrhea with the presence of methane (1-way ANOVA); p <0.05 for the tendency towards increased constipation with the presence of methane (1-way ANOVA).

Figure 8 shows the severity classifications of diarrhea and average constipation of IBS subjects (n = 296) with SIBO as a function of the type of gas pattern produced in LBT; p <0.001 for tendency to reduce diarrhea with the presence of methane (1-way ANOVA); p <0.05 for the tendency towards increased constipation with the presence of methane (1-way ANOVA).

Figure 9 shows the classification of severity of mean constipation minus diarrhea (C-D) for the entire group (n = 551) and IBS subjects (n = 296) as a function of the type of gas pattern produced in LBT; p <0.00001 for C-D trend for the entire group (one-way ANOVA); p <0.0001 for C-D trend for IBS subjects (one-way ANOVA).

Figure 10 shows the percentage of IBS subjects (n = 296) exhibited by each gas pattern who report predominant symptoms of constipation vs. diarrhea.

Figure 11 shows the percentage of subjects with IBD who produce each of the three abnormal gas patterns in LBT.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

A first aspect of the invention provides a selective methanogenic inhibitor, selected from monensin and an HMG-CoA reductase inhibitor, for use in the treatment of constipation.

A further aspect of the invention provides an agent comprising methane or a methane precursor that causes the partial pressure of methane in the intestine of a subject that has been treated to be increased for use in the treatment of diarrhea. An embodiment of this aspect of the invention is where the agent is a methane precursor comprising a catalyst and chemical substrate that causes the partial pressure of methane in the intestine of a subject that has been treated to increase.

According to the use of the claimed invention, the partial pressure of methane in the intestine of the subject can be reduced by administering to the intestinal lumen of the subject a selective inhibitor of methanogeny, such as monensin. Useful selective inhibitors of methanogeny include the HMG-CoA reductase inhibitors known in the art (e.g., U.S. Patent No. 5,985,907) that selectively inhibit the growth of methanogenic bacteria without significantly inhibiting the growth of non-methanogens, for example in the intestine distal or colon of the subject.

Alternatively, the partial pressure of methane in the subject's intestine can be reduced by administering to the subject's intestinal lumen the probiotic agent that displaces the methanogen to inhibit the growth of methanogenic bacteria there, for example, an inoculum of a lactic acid bacterium, bifidobacteria, or probiotic Saccharomyces species, for example, S. cerevisae. (AS. Naidu et al., Probiotic spectra of lactic acid bacteria, Crit. Rev. Food Sci. Nutr. 39 (1): 13-126 [1999]; J, A. Vanderhoof et al. [1998]; GW Tannock , Probiotic propertyies of lactic acid bacteria: plenty of scope for.R. & D, Trends Biotechnol. 15 (7): 270-74 [1997]; S. Salminen et al., Clinical uses of probiotics for stabilizing the gut mucosal barrier : successful strains and future challenges, Antonie Van Leeuwenhoek 70 (2-4): 347-58 [1997]; Chaucheyras F. et al., In vitro H2 utilization by a ruminal acetogenic bacterium cultivated alone or in association with an archaea methanogen is stimulated by a probiotic strain of Saccharomyces cerevisiae, Appl Environ Microbiol 61 (9): 3466-7 [1995]). The inoculum is typically administered in a pharmaceutically acceptable ingestible formulation, such as in a capsule, or for some subjects, it is effective by consuming a food supplemented with the inoculum, for example milk, yogurt, cheese, meat or other fermentable food preparation. Useful prebiotic agents include Bifidobacterium sp. or Lactobacillus species or strains, for example, L. acidophi / us, L. rhamnosus, L. plantarum, L reuteri, L paracasei subsp, parracasei, or L. casei Shirota, (P. Kontula et al., The effect of lactose derivatives on intestinal lactic acid bacteria, J. Dairy Sci. 82 (2): 249-56 [1999]; M. Alander et al, The effect of probiotic strains on the microbiota of the Simulator of the Human Intestinal Microbial Ecosystem (SHlME) Int. J. Food Microbiol 46 (1): 71-79 [1999]; S. Spanhaak et al., The effect of consumption of milk fermented by Lactobacillus casei strain Shirota on the intestinal microflora and immune parameters in humans, Eur.

J. Clin. Nutr. 52 (12): 899-907 [1998]; W.P. Charteris et al., Antibiotic susceptibility of potentially probiotic Lactobacillus species, J. Food Prot. 61 (12): 1636-43 [1998]; B.W. Wolf et al., Safety and tolerance of Lactobacillus reuteri supplementation to a population infected with the human immunodeficiency virus, Food Chem. Toxicol, 36 (12): 108594 [1998]; G. Gardiner et al., Development of a probiotic cheddar cheese containing human-derived Lactobacillus paracasei strains, Appl. Environ. Microbiol 64 (6): 2192-99 [1998]; T. Sameshima et al., Effect of intestinal Lactobacillus starter cultures on the behavior of Staphylococcus aureus in fermented sausage, Int. J. Food Microbiol 41 (1): 1-7 [1998]).

Alternatively, the partial pressure of methane in the subject's intestine can be reduced by administering to the subject's intestinal lumen the prebiotic agent that inhibits the growth of methanogenic bacteria or promotes the growth of competent non-methanogenic intestinal flora. (For example, Tuohy KM et al., The prebiotic effects of biscuits containing partially hydrolysed guar gum and fructo-oligosaccharides-a human volunteer study, Br J Nutr 86 (3): 341-8 [2001]).

According to the use of the claimed invention, the partial pressure of methane in the subject's intestine can be increased by administering methane to the subject's intestinal lumen. According to the above, methane can be administered directly to the intestine through a tube, preferably through the rectum, but other access routes for intubation to the intestine are also useful. Alternatively, methane can be administered to the intestinal lumen by providing a medicament comprising a catalyst and chemical substrate (ie, a "methane precursor") to the intestinal lumen, where they come into contact to produce methane in situ. For example, the catalyst and the substrate can be administered in separate controlled-release tablets, which release their contents at the desired location in the intestine.

Alternatively, the partial pressure of methane in the subject's intestine can be increased by administering to the subject's intestinal lumen the prebiotic agent that improves methane. A probiotic agent "that improves methane" is one that effectively improves the partial pressure of methane in the subject's intestinal lumen. The prebiotic agent that improves methane can be a methanogenic bacterium, such as Methanobrevibacter smithii, or certain Bacteroides spp, or Clostridium spp. (See, for example, McKay LF et al., Methane and hydrogen production by human intestinal anaerobic bacteria, Acta Pathol Microbiol Immunol Scand [B] 90 (3): 257-60 [1982]), or an organism that can improve the growth of intestinal methanogens, such as Clostridium butiricum.

Alternatively, the partial pressure of methane in the subject's intestine can be increased by administering to the subject's intestinal lumen a prebiotic agent that improves the growth of methanogenic bacteria.

As the term is commonly used in the art, the "proximal" segment of the small intestine, or "proximal intestine," comprises approximately the first half of the small intestine of the pylorus to the middle part of the intestine. The distal segment, or "distal intestine" includes approximately the second half, from the middle intestine to the ileocecal valve.

Representative methods for administration include giving, providing, feeding or feeding by force, dispensing, inserting, injecting, infusing, perfusing, prescribing, presenting, dealing with, taking, swallowing, swallowing, eating or applying. The administration of inhibitors, probiotic agents, or prebiotic agents, is by well known means, which more preferably include oral administration and / or enteric administration.

The detection of intestinal methane and other gases, although not essential for the practice of the invention, can be carried out, if desired, by any suitable means or methods known in the art. For example, a preferred method is the breath test. (For example, P. Kerlin and L. Wong, Breath hydrogen testing in bacterial overgrowth of the small intestine, Gastroenterol. 95 (4): 982-88 [1988]; A. Strocchi et al., Detection of malabsorption of low doses of carbohydrate: accuracy of various breath H2 criteria, Gastroenterol. 105 (5): 1404-1410 [1993]; D. de Boissieu et al., [1996]; PJ Lewindon et al., Bowel dysfunction in cystic fibrosis: importance of breath testing, J. Paedatr. Child Health 34 (1): 79-82 [1998]). The breath hydrogen or methane breath tests are based on the fact that many facultatively or obligatorily fermentative bacteria found in the gastrointestinal tract produce detectable amounts of hydrogen or methane gas as fermentation products of a substrate consumed by the host, under certain circumstances. Substrates include sugars such as lactulose, xylose, lactose, sucrose, or glucose. Hydrogen or methane produced in the small intestine that enters the host's bloodstream and exhales gradually.

Typically, after fasting overnight, the patient swallows a controlled amount of a sugar, such as lactulose, xylose, lactose, or glucose, and breath samples are taken at frequent time intervals, typically every 10 to 15 minutes during a four hour period. The samples are analyzed by gas chromatography

or by any other suitable techniques, alone or in combination. A variable fraction of the population fails to exhale appreciable hydrogen gas during the intestinal fermentation of lactulose; The intestinal microflora of these individuals instead produces more methane. (G. Corazza et al., Prevalence and consistency of low breath H2 excretion following lactulose ingestion. Possible implications for the clinical use of the H2 breath test, Dig. Dis. Sci. 38 (11): 2010-16 [1993]; SM Riordan et al., The lactulose breath hydrogen test and small intestinal bacterial overgrowth, Am. J. Gastroentrol. 91 (9); 1795-1803 [1996]). Optionally, the non-digestible substrate other than lactulose can be used.

Another useful method for detecting intestinal gases, such as methane, is by gas chromatography with mass spectrometry and / or radiation detection to measure carbon dioxide emissions labeled with isotope, methane, or hydrogen, after administering a substrate. Isotope labeling that is metabolizable by gastrointestinal bacteria but poorly digestible by the human host, such as lactulose, xylose, mannitol, or urea. (For example, G.R. Swart and J.W. van den Berg, 13C breath test in gastrointestinal practice, Scand.

J. Gastroenterol. [Suppl.] 225: 13-18 [1998]; S.F. Dellert et al., The 13C-xylose breath test for the diagnosis of small bowel bacterial overgrowth in children, J. Pediatr. Gastroenterol Nutr. 25 (2): 153-58 [1997]; EC. King and P.P. Toskes, Breath tests in the diagnosis of small intestinal bacterial overgrowth, Crit. Rev. Lab. Sci. 21 (3): 269-81 (1984]). A poorly digestible substrate is one for which there is a relative or absolute lack of capacity in a human for absorption or for enzymatic degradation or catabolism.

Suitable isotopic marks include 13C or 14C. To measure methane or carbon dioxide, suitable isotopic tags may also include 2H and 3H or 17O and 18O, while the substrate is synthesized with the isotopic tag placed in a metabolically appropriate location in the substrate structure, that is, a location where enzymatic biodegradation by intestinal microflora results in the isotopic mark that is sequestered in the gaseous product. If the selected isotopic mark is a radioisotope, such as 14C, 3H, or 15O, breath samples can be analyzed by gas chromatography with suitable radiation detection means (For example, CS Chang et al., Increased accuracy of the carbon -14 D-xylose breath test in detecting small intestinal bacterial overgrowth by correction with the gastric emptying rate, Eur. J. Nucl. Med. 22 (10): 1118-22 [1995]; CE King and PP Toskes, Comparison of the 1-gram [14C] xylose, 10-gram lactulose-H2, and 80-gram glucoseH2 breath tests in patients with small intestinal intestine overgrowth, Gastroenterol. 91 (6): 1447-51 [1986]; A. Schneider et al. , Value of the 14C-D-xylose breath test in patients with intestine / bacterial overgrowth, Digestion. 32 (2): 86-911 [1985]).

The invention will now be described in more detail with reference to the following non-limiting examples.

EXAMPLES

Example 1. Methane excretion in breath test has a positive predictive value of 100% for constipation-predominant IBS.

In a randomized, double-blind, placebo-controlled study, we confirm that a lactulose breath test

5 Abnormal is more prevalent in IBS than normal controls, and that antibiotic treatment in IBS leads to an improvement in symptoms and that this is based on the antibiotic-induced normalization of the breath test. Second, lactulose breath test profiles are evaluated by showing that gaseous constituents vary between IBS subgroups. In this study, we show that methane excretion in the breath test has a positive predictive value of 100% for predominant IBS constipation. This is another important stage in the binding of SIBO and IBS.

A. Materials and Methods

Study population

Study subjects are linked through advertisements in local newspapers, radio and IBIS support groups throughout most of the Los Angeles area. To avoid reference bias, subjects are not linked to

15 through the clinical motility of GI or any gastroenterological practice based at the Cedars-Sinai Medical Center. Subjects are included if they meet the Rome I criteria for IBS (7). Roma I is selected when it does not differentiate between diarrhea and constipation, and peer review publications are not available to validate Roma II as a diagnostic strategy (14). Subjects are excluded if they have antibiotics within the previous three months, a previous lactulose breath test (LBT), or a history of diabetes, thyroid disease, bowel surgery (except cholecystectomy or appendectomy), connective tissue disease, Narcotic use or known gastrointestinal disease. Subjects with renal insufficiency, cardiac deterioration, probiotic use or allergy to aminoglycosides are also included. The approval of the institutional review board and informed written consent of the participating subjects is obtained.

In an initial comparison, 15 normal controls matched by sex are identified based on the absence of

25 all Rome I criteria. These subjects underwent the lactulose breath test and the prevalence of abnormal breath test is compared with subjects with IBS.

Study design

Subjects submitted to the GI Motility Laboratory have a 7:00 p.m. fast. From the previous night. They are not instructed to ingest legumes or a heavy meal for dinner the night before evaluation. Good oral hygiene is recommended and smoking is not allowed on the day of the test.

Before the LBT, the subjects complete a symptom questionnaire that asks them the degree of nine IBS symptoms (abdominal pain, diarrhea, constipation, inflammation, sense of incomplete evacuation, defecation effort, rectal tenesmus, mucus and gas) in a classification of severity of 0-5 as previously used and recommended (15-17). All questions are asked based on your memory of the previous 7 days (17).

35 The subjects then experienced a LBT by ingesting 10g of lactulose (Inalco Spa, Milano, Italy, packaged by Xactdose Inc., South Beloit, IL) followed by 1-2 ounces of water after an initial initial breath sample. Breath samples are then collected at 15 minute intervals for 180 minutes. Final expiratory breath samples are taken to secure the alveolar gas sample. Samples are analyzed for hydrogen, methane and carbon dioxide using a Quintron Model SC gas chromatography (Quintron Instrument Company, Milwaukee, WI). Carbon dioxide measurements are used to correct the quality of the alveolar sample. The measurements are drawn graphically as previously described (12). The patients and researchers were blinded for the breath test result.

All subjects are randomized by staff not associated with the study to receive, in a double-blind form, neomycin (500 mg) (Teva pharmaceuticals, USA, Sellersville, PA) or placebo for equalization twice 45 daily for 10 days. Seven days after the termination of the antibiotic or placebo, the subjects return for a repeated questionnaire and LBT. One day of the next seven days is selected because in our experience the IBS abnormal breath test can recur as soon as two weeks after the antibiotic normalization. As part of the following questionnaire, subjects are asked to subjectively classify the amount of improvement they experience as a percentage of normalization of bowel function and repeat their perceived severity of the 9 bowel symptoms described above. Compliance is assessed by pill counting. To meet the requirements of the institutional review board,

The results of the LBT follow-up cannot be blinded in such a way that patients can seek appropriate medical therapy for their test result.

Upon termination of binding, all initial and subsequent breath tests are coded and randomized by personnel not involved in the interpretation of the test. A blinded reviewer (M.P.) interprets the results and is questioned to categorize the breath tests based on whether the test meets the criteria for normal LBT. A normal LBT is defined, without increasing the concentration of hydrogen (H2) or methane (CH4) in breath before 90 minutes of lactulose, with a definitive increase of more than 20 ppm during 180 minutes of measurement (18, 19, 37 , 38). Studies that escape this range are categorized as abnormal. A second set of criteria is required for the interpretation of the breath test also therefore uses the 2 traditional peaks to suggest bacterial overgrowth. Because the two-spike method is not as good as the one that validates a technique (37) such as parts per million (ppm), these findings are only used to compare the prevalence of this finding for healthy controls.

Result Measurements

Data are analyzed using an intention to treat method. The primary outcome measurement is based on a composite classification (CS) calculated from the 3 main IBS symptoms (abdominal pain, diarrhea and constipation each on a scale of 0-5) to generate a classification of 15 (more severe). This is done to count the severity of all potential IBS subgroups. Because other IBS symptoms (such as defecation effort) would improve or worsen depending on whether patients start with diarrhea or constipation, respectively, minor criteria are not included in the CS. Additionally, as the reduction in colonic organisms can result in an improvement in gas and inflammation, independent of ascending bacterial growth, very gaseous symptoms are excluded from the classification. The percentage improvement in CS between placebo and neomycin is then compared. Additionally, the general percentage of normalization of the intestine is determined according to the patient's report and compared in a similar way.

The prevalence of a true clinical response is then determined and compared between placebo and neomycin. A true clinical response is defined as a ≥ 50% reduction in CS. Secondly, a true clinical response is also evaluated based on the subjects who report their general percentage of bowel normalization. A normalization of ≥ 50% implies a true clinical response. This method of analysis closely follows the multinational consensus that recommends guidelines for data analysis in IBS clinical studies (16).

Secondary endpoints include a similar analysis of gender subgroups. Subsequently, the IBS subgroups are identified so that the predominant IBS diarrhea is considered present when the severity of the diarrhea (0-5 scale) is greater than constipation in any individual subject. The opposite proportion determines the predominance of constipation. These means of identifying the predominant diarrhea and constipation subgroups are selected because the criteria for these subgroups are not validated and subjectively based on the interview with the doctor (14). This method further reduces bias because the subjects would not be aware of the interest in their predominant characteristic of the subgroup.

A post hoc analysis is then conducted on all abnormal breath test results to determine if the type of gas produced in LBT is related to the IBS subgroup. Abnormal breath tests are divided into two abnormal test groups: hydrogen production only and any methane production. The relationship between predominant IBS constipation and predominant IBS diarrhea is determined for the type of gas seen. Subsequently, in a more objective way, the classification of severity for diarrhea and constipation is then compared between the types of gas. Finally, a classification is determined based on the difference between the severity of constipation and diarrhea (ie, constipation classification minus diarrhea classification; "C-D"). C-D is used to examine the relative weight of constipation to diarrhea in individual subjects (the most positive classification of greater constipation domain is compared to diarrhea). Subjects with identical classification by severity of constipation and diarrhea are excluded from these analyzes. This C-D classification is also compared with the types of gas.

Finally, to support the main test that the abnormal in IBS is not due to rapid transit, the test profile of medium breath in constipation and predominant IBS diarrhea is compared. Because it is suggested in the literature that predominant IBS diarrhea is associated with rapid transit (34-36) and predominantly IBS constipation with slow transit (34, 35), the hydrogen profile should be different in both groups.

Statistic analysis

The number of subjects linked in the study is determined based on the detection of a 10% difference between placebo and neomycin. This additionally assumes a variance of 15% and an α = 0.05 with power of 90% in a 2-sided analysis.

Quantitative data are compared using the Student's t-test with results expressed as mean D.E. Comparisons of qualitative data use Fisher's Exact Test for the comparison of IBS subjects with healthy controls. All other comparisons of qualitative data use Chi-square. A 1-way ANOVA is used to compare the results of the 3 groups: placebo treated, non-normalized neomycin

5 successful LBT and neomycin treated with successful normalization of LBT.

B. Results

Demographics of the subjects

Two hundred and thirty one subjects are detected (Figure 1). Of these, 111 meet the criteria for involvement. However, 10 of these 111 subjects have incomplete data (6 in the neomycin group and 4 in the placebo group).

10 The specific reasons for incomplete data are, voluntary premature withdrawal (n = 3), does not follow the breath test (n = 4), fails to show up for follow-up (n = 1), does not follow the questionnaire (n = 1 ) and premature withdrawal by the subject due to severe diarrhea (n = 1). Despite incomplete data, these subjects are included in the intention to treat analysis and they are counted as no improvement (0%). The characteristics of initial values are similar for the neomycin and placebo groups (Table 1, below).

15 Table 1. Demographic comparison between placebo and neomycin.

Feature Placebo Neomycin Value p

N 56 55 Age 41.9 ± 0.2 44.7 ± 0.2 NS Sex (F / M) 27/29 34/21 NS Composite classification of initial values 8.7 ± 0.4 8.8 ± 0.3 NS Abnormal breath test [n (%)] 47 (84) 46 (84) NS Predominant IBS diarrhea [n (%)] 21 (40)> 25 (48) * NS Predominant IBS constipation [n (%)] 20 (38)> 18 (35) * NS Other IBS subgroup [n (%)] 11 (21)> 7 (13) * NS

The data are initial values of mean ± D.E. classification compounds = severity of pain + classification of diarrhea + classification of constipation (each on a scale of 0-5) before treatment. Other subgroups IBS = subjects with severity of constipation = severity of diarrhea.

* Only 52 subjects in the neomycin group completed the questionnaire sufficiently to determine this result.

> Only 52 subjects in the placebo group completed the questionnaire sufficiently to determine this result. NS = not significant.

Case Control Comparison

IBS subjects have a higher prevalence of abnormal LBT than controls matched by sex with 93 of 111 (84%) subjects who meet these criteria compared to 3 of 15 (20%) controls matched by sex (OR = 26.2, CI = 4.7-103.9, p <0.00001). When comparing the prevalence of abnormal LBT with double peak, 55 of 111

20 IBS subjects (50%) are positive compared to 2 of 15 healthy controls (13%) (p = 001).

Primary Outcome Measurements

In the intention-to-treat analysis, neomycin results in a reduction of 35.0 ± 0.7% in CS compared to a reduction of 11.4 ± 1.3% in the placebo group (p <0.05). In the subgroup of patients with LBT of abnormal initial values (n = 93), neomycin produces a reduction of 35.4 ± 0.8% in CS versus a reduction of

25 3.7 ± 1.6% in the placebo group (p <0.01). No difference is seen in subjects with normal initial values breath test although a higher placebo index is reported in this very small group (51%).

Ninety-one of the 111 subjects completed their percentage of bowel normalization questions after treatment. Of these 91 subjects, neomycin results in a reported bowel normalization of 40.1 ± 5.3% compared to 15.1 ± 3.6% for placebo (p <0.001). Among the subgroup of subjects with abnormal initial breath tests, neomycin results in a normalization of 44.8 ± 5.6% compared to 11.0 ± 3.3% for placebo (p <0.00001).

Neomycin results more easily in true clinical response than placebo. Among all subjects who received neomycin, 24 of 55 (43%) experienced an improvement of ≥ 50% in CS versus 13 of 56 (23%) in the placebo group (OR = 4.3, CI = 1.05-6.3, p < 0.05). In the subgroup of subjects with abnormal breath tests, 21 of 46 (46%) received neomycin that has a clinical response compared to 7 of 47 (15%) in the placebo group (OR = 4.8, CI = 1.62-14.7 , p <0.01). Using the objective report of the patients of the percentage of normalization of the intestine, in the complete group of subjects who answered this question (n = 91), 50% of the subjects who received neomycin had a true clinical response in contrast to 17% of the subjects who obtained placebo (OR = 4.8, CI = 1.7-14.4, p <0.01). In those with abnormal initial breath test, subjects treated with 55% neomycin and 11% placebo have a true clinical response (OR = 9.6, CI = 2.5-39.7, p <0.0001). Finally, 7 of the 8 subjects (88%) who have normal LBT after reported neomycin have more than 50% normalization of bowel function.

Of the 111 subjects, only the 101 subjects with complete data are used in the rest of the analysis.

Of 84 of the 101 subjects with an abnormal initial LBT, 41 are treated with neomycin. Eight of 41 (20%) reached the normalization of the LBT. One of the 43 subjects in the placebo group was on an abnormal to normal breath test. A significant difference in the response to the symptom is seen depending on the outcome of the treatment in these abnormal subjects. Specifically, the percentage reduction in CS is different in the following 3 groups: subjects receiving placebo (4.1 ± 11.7%), group treated by neomycin that does not reach LBT normalization (34.4 ± 6.2%) and the group treated with neomycin with LBT normalization (61.7 ± 9.4%) (p = 0.01, one-way ANOVA) (Figure 2). Using self-reported patients of the percentage of normalization of the intestine, the 3 groups are more different. Subjects receiving placebo report 11.0 ± 3.7% normalization, subjects receiving neomycin but not successful normalization of LBT, 36.7 ± 6.1% and those subjects with normal LBT follow-up after neomycin report 75.0 ± 6.4% of bowel normalization ( p <0.0000001, 1-way ANOVA).

Neomycin, although statistically more effective than placebo, is only able to normalize the breath test 20% of the time. This may be due to large numbers and types of enteric organisms (30-33) or bacterial resistance.

Transit comparison

When comparing the average hydrogen breath test among subjects with diarrhea and predominant IBS constipation, there is no evidence that the predominance of diarrhea has an earlier occurrence of hydrogen (data not shown). In fact, the profiles of diarrhea and constipation are both virtually superimposed and are not different at any time point with an average of> 20 ppm in 90 minutes in both groups.

Adverse events

A subject develops profuse watery diarrhea while taking placebo. Then it is found that the origin of diarrhea is from food poisoning. It is found that two of the subjects involved have other diagnoses. The first subject has a mass of 8 cm in the abdomen. The surgical specimen demonstrates non-Hodgkin lymphoma. This subject is in the placebo group. It is observed that the second subject has urinary retention, which precipitates complications to the intestine. The second subject is in the neomycin group. These subjects have a normal initial LBT. Both are included as part of the intention to treat analysis.

Gender effect

It is observed that both male and female subjects have a significantly greater improvement in the percentage of normalization of the intestine over placebo (Figure 3). Additionally, there is no difference in the response rate between male and female patients.

Type of gas and IBS subgroup

The type of gas produced by IBS subjects in LBT is predictive of their IBS subtype among the 84 subjects with abnormal initial values. After exclusion of subjects without gas production (n = 4) and subjects where the severity of constipation is equal to diarrhea (n = 15), 34 subjects with predominant diarrhea and 31 with constipation and predominant IBS are analyzed . Twenty of the 31 subjects with predominant constipation (39%) excreted methane while no methane excretion is seen in the 34 subjects with predominant diarrhea (OR = ∞, CI = 3.7-4.3,

p <0.001, positive predictive value = 100%) (Table 2, below; and Figure 4). The severity of constipation is 4.1 ± 0.3 in subjects with methane excretion but only 2.3 ± 0.2 in excretors without methane (p <0.01) (Table 3, below). In a similar comparison, C-D is 2.8 ± 0.5 in methane excretors and -0.7 ± 0.3 for hydrogen excretors (p <0.00001) (Table 3; and see Figure 5).

Table 2: Comparison of IBS subgroups based on methane and hydrogen excretion with abnormal breath test.

Methane hydrogen

(n = 65) * Diarrhea 34 0 Constipation 19 12

* After exclusion of subjects without gas production (n = 4), normal breath test (n = 17) and subjects where diarrhea severity = constipation severity (i.e. not predominant) (n = 15 ). P <0.001 between groups.

Table 3. Evaluation of the severity of constipation or diarrhea based on methane production in the initial values breath test.

Methane free Methane P-value

Severity of 2.3 ± 0.2 4.1 ± 0.3 <0.001 constipation Severity of diarrhea 3.0 ± 0.2 1.4 ± 0.4 <0.001 C-D classification * -0.7 ± 0.3 2.8 ± 0.5 <0.00001

* The C-D classification represents the difference between the severity of constipation and diarrhea. This is done to show an increased relative weight of constipation to diarrhea with methane excretors.

10

Regardless of any argument about whether the breath test reliably detects SIBO or not, the data in this study support a role of the LBT in the treatment of IBS since only when the subsequent LBT is normal that the greatest symptom improvements are made .

Although the discussion thus focused on the breath test of abnormal intestinal flora, it is necessary to discuss another

15 possible interpretation. The abnormal breath tests seen in this study may represent rapid transit. Studies have suggested that the transit of the small intestine is accelerated in the predominant IBS diarrhea (34-36). Similar studies suggest that subjects with predominant IBS constipation have delayed transit (34, 35). If transit is the explanation for abnormal breath test findings then subjects with predominant IBS constipation should have delayed gas that arises in the breath test. For him

On the contrary, in our study, breath tests are abnormal independent of the IBS subgroup that suggests that transit alone cannot explain the findings. Additionally, clinical improvement in a composite classification (consisting of diarrhea, constipation and abdominal pain) that depends on the normalization of LBT cannot be explained on the basis of transit alone.

In summary, in this randomized, double-blind, placebo-controlled study, we found a higher prevalence of

25 Abnormal lactulose breath tests in IBS patients that controls, indicative of SIBO. Additionally, we find that antibiotics are more effective than placebo in terms of symptom improvement and the normalization of the breath test results in an even greater improvement of IBS symptoms, resulting substantially from a previous study (12). Additionally, we found that methane excretion in the breath test is largely associated with the predominant constipation subset of IBS. The ability to

30 Identifying IBS subgroups based on LBT additionally supports the association between SIBO and IBIS. The presence of SIBO in IBIS patients is consistent with the existence of persistent antigenic exposure in IBS.

Example 2. Administration of methane to the distal intestine slows gastrointestinal transit.

We now show that methane administered directly to the distal intestine causes a decrease in gastrointestinal transit. In dogs equipped with the duodenum (10 cm of the pylorus) and middle intestine (160 cm of the pylorus) fistulas, intestinal transit is compared through an isolated 150 cm test segment (between the fistulas)

5 while the proximal segment of the intestine has perfusion with phosphate buffer at pH 7.0 at 2 mL / min for 90 minutes. Ambient air in = three dogs) or methane (n = three dogs) is supplied in the distal intestine as a 180-ml bolus at time 0. Sixty minutes after the start of infusion, 20 µCi of 99mTc- DTPA (diethylenetriaminepentaacetic acid) It is supplied as a bolus in the proximal segment of the intestine. The intestinal transit is then measured by contacting the 1 ml samples collected every 5 minutes after the diversion of the fistula from the middle intestine in radioactivity.

Intestinal transit is calculated by determining the area under the curve (AUC) of the percentage of cumulative recovery of the radioactive marker in control (air administration) and experimental (methane administration) dogs. The square root values of the AUC (Sqrt AUC), where 0 = no recovery for 3 0 minutes and 47.4 = theoretical, the instantaneous full recovery at time 0, are compared with the control and experimental animals,

15 using two-way ANOVA for repeated measurements.

The results shown in Figure 6 demonstrate that administration of methane to the distal intestine substantially decreases the rate of intestinal transit in the experimental group, compared to the control.

Example 3

The following study further confirms and investigates the relationship between gastrointestinal complications (specifically, diarrhea and constipation) in subjects diagnosed with IBS with SIBO and gas excretion in LBT in a prospectively larger database. The prevalence of gas excretion patterns in IBS and predominantly diarrheal conditions of Crohn's disease and ulcerative colitis are also compared.

A. Materials and Methods.

Patient Population

25 Consecutive patients referred for a lactulose breath test (LBT) by the Cedars-Sinai Medical Center, 1998-2000 GI Motility Program complete a questionnaire designed to assess bowel symptoms as previously described (12) after of the approval of the institutional review board. Subjects are asked for the severity rate of nine symptoms (diarrhea, constipation, abdominal pain, inflammation, feeling of incomplete evacuation, defecation effort, incontinence, mucus, and gas) on a scale of 0-5, 0 which means absence of the symptom The questionnaire also infers if the subjects have Crohn's disease (CD)

or ulcerative colitis (UC). Of the subjects who report a history of inflammatory bowel disease (IBD), only those whose diagnosis has been confirmed by the CedarsSinai Inflammatory Bowel Disease Center are included in the analysis. The diagnosis of the IBS identifies whether the subjects meet the Rome I criteria (7). It is found that subjects who have IBD and IBIS are assigned to the IBD subgroup.

35 subjects with conditions predisposed to rapid transit (small intestine syndrome, gastrectomy, etc.), those taking narcotic medications, and those without evidence of ascending overgrowth of LBT are excluded.

Lactulose breath test (LBT)

After an overnight fast, the subjects complete the questionnaire. A breath sample of initial values is then obtained after which the subjects ingest 10 g of lactulose syrup (Inalco Spa, Milano, Italy, packaged by Xactdose Inc., South Beloit, IL). This is followed by 1 ounce of sterile water. Breath samples are then collected every 15 minutes for 180 minutes. Each sample is analyzed for concentration of hydrogen, methane, and carbon dioxide within 15 minutes of collection using Quintron Model SC gas chromatography (Quintron Instrument Company, Milwaukee, WI.) CO2 is analyzed to correct the quality of the

45 alveolar sample.

Three different patterns of abnormal gas are described after the end of the test:

one.

Positive hydrogen breath test: rises in hydrogen concentration in breath> 20ppm within 90 minutes of ingestion of lactulose (18, 19, 37, 38).

2.

Positive breath test for hydrogen and methane: rises in both concentrations of hydrogen and methane in breath of> 20 ppm within 90 minutes of ingestion of lactulose.

3.

 Methane positive breath test: It rises in the concentration of methane in breath of> 20ppm within 90 minutes of ingestion of lactulose.

Analysis of data

For all subjects with SIBO, classifications of diarrhea severity and mean constipation 5 are compared among the three abnormal gas patterns.

Based on the severity classifications of the symptom, the entire IBS group is further subdivided into the predominant subgroups of diarrhea and predominant constipation. The predominant IBS constipation is identified if a subject severity classification exceeds its diarrhea severity classification, while the inverse for the predominant IBS diarrhea is applied. Subjects who have a severity classification

10 constipation equal to the classification severity of diarrhea (indeterminate pattern) are excluded from the IBS subgroup analysis. The percentage of IBS subjects within each abnormal gas pattern who report the symptoms of predominant constipation or predominant diarrhea is tabulated. The prevalence of methane production among IBS subgroups is also compared.

Subsequently, an average C-D classification is obtained by calculating the difference between severity classifications

15 constipation and diarrhea. This is used to examine the relative weight of constipation to diarrhea in individual subjects. The C-D classification is compared between the three abnormal breath gas patterns in the group as a whole and among IBS subjects.

Finally, the prevalence of each of the three abnormal gas patterns is evaluated in subjects with CD and UC. The prevalence of methane production is contrasted among subjects with IBS and IBS.

20 Statistical Analysis

A one-way ANOVA is conducted to compare symptom severity ratings among the three gas patterns in LBT. Prevalence data are analyzed with a chi-square test.

B. Results:

Subjects

25 At the time of the analysis, the 772 patients are referred for LBT and are entered into the database. One hundred eighty-three subjects with a negative breath test, and 38 subjects taking narcotic medications or with conditions predisposed for rapid transit are excluded. A total of 551 subjects remain for analysis. Of these, 78 carry the diagnosis of IBD (49 with CD and 29 with UC) and 296 without IBD meet the Rome I criteria for IBS. Of the subjects with IBS, 120 report symptoms of predominant constipation, 111 have diarrhea symptoms

30 predominant, and 65 have a classification of severity of constipation equal to the classification of severity of diarrhea.

Bacterial Ascending Growth Analysis

When evaluating the entire group of subjects with SIBO (n = 551), the diarrhea severity classifications differ significantly between the three abnormal breath test patterns (one-way ANOVA, p <0.00001)

35 (Figure 7). Subjects who excrete methane report significantly lower diarrhea severity classifications than those who only produce hydrogen. The severity of constipation also differs significantly between breath test patterns (p <0.05), with higher severity ratings reported by subjects who produce methane.

Among all IBS subjects (n = 296), diarrhea severity classifications also differ similarly.

40 (one-way ANOVA, p <0.001) with seventy lower reported by those who produce methane than those who produce only hydrogen gas (Figure 8).

When the C-D classification is evaluated as a reflection of the degree of constipation with respect to diarrhea, the effect of methane is even more obvious (Figure 9). In the total group and IBS subjects, constipation is the most prevalent symptoms in individuals, while diarrhea is the prevalent symptom of subjects with only hydrogen.

45 When the IBS subgroups are compared, predominant IBS constipation is reported by 91 (37%) of the subjects that excrete hydrogen, 23 (52.3%) of the subjects excrete hydrogen and methane and 6 (100%) of the subjects excrete methane. In contrast, predominant IBS diarrhea is observed in 105 (42.7%) of hydrogen excretors, 6 (13.6%) of hydrogen and methane excretors, and none of methane excretors (Figure 10).

Inflammatory bowel and methane disease

The predominant gas excreted by patients with IBD is only hydrogen, detected in 47 of the 49 subjects (95.9%) with Crohn's disease and 29 of the 29 (100%) subjects (100%) with ulcerative colitis. (Figure 11).

Methane production among subjects with IBS and IBD

5 The percentage of subjects with IBS who produce each of the three gas patterns is tabulated. Of the 296 IBS subjects, 246 (83.1%) produce only hydrogen gas, 44 (14.9%) produce hydrogen and methane gas, and 6 (2.0%) produce only methane gas. Methane production depends significantly on whether or not the subjects have IBS or IBD. IBS subjects produce methane gases more easily than subjects with ulcerative colitis or Crohn's disease (OR 7.7, CI 1.8 - 47.0, p <0.01) (Table 4).

10 Table 4. Comparison of the prevalence of methane to non-methane gas production among subjects with IBS and IBD.

Type of disease CH4 Without CH4

IBS (n = 296) 50 246 UCorCD (n = 82) 2 76

Chi square 9.4, OR 7.7, CI 1.8 - 47.0, p value <0.01

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Claims (3)

one.

A selective methanogenic inhibitor, selected from monensin and an HMG-CoA reductase inhibitor, for use in the treatment of constipation.

2.

An agent comprising methane or a methane precursor that causes the partial pressure of methane in the intestine of a subject that has been treated to be increased for use in the treatment of diarrhea.

3.

The agent according to claim 2 for use in the treatment of diarrhea wherein the agent is a methane precursor comprising a catalyst and chemical substrate that causes the partial pressure of methane in the intestine of a subject that has been treated. To increase.